Sex-specific effects of a high fat diet on aortic inflammation and dysfunction

Obesity and vascular dysfunction are independent and sexually dimorphic risk factors for cardiovascular disease. A high fat diet (HFD) is often used to model obesity in mice, but the sex-specific effects of this diet on aortic inflammation and function are unclear. Therefore, we characterized the aortic immune cell profile and function in 6-week-old male and female C57BL/6 mice fed a normal chow diet (NCD) or HFD for 10 weeks. Metabolic parameters were measured weekly and fortnightly. At end point, aortic immune cell populations and endothelial function were characterized using flow cytometry and wire myography. HFD-male mice had higher bodyweight, blood cholesterol, fasting blood glucose and plasma insulin levels than NCD mice (P < 0.05). HFD did not alter systolic blood pressure (SBP), glycated hemoglobin or blood triglycerides in either sex. HFD-females had delayed increases in bodyweight with a transient increase in fasting blood glucose at week 8 (P < 0.05). Flow cytometry revealed fewer proinflammatory aortic monocytes in females fed a HFD compared to NCD. HFD did not affect aortic leukocyte populations in males. Conversely, HFD impaired endothelium-dependent vasorelaxation, but only in males. Overall, this highlights biological sex as a key factor determining vascular disease severity in HFD-fed mice.


Blood pressure measurement
Systolic BP (SBP) was measured weekly by a non-invasive tail cuff plethysmography (MC4000 Multi Channel Blood Pressure Analysis System, Hatteras Instruments, Cary, NC, USA) as previously described 28 .Mice were acclimated 1 week prior to BP measurements to minimize stress.Mice underwent 10 acclimation cycles, followed by 4 sets of 15 cycles.The data output was recorded using MC4000 Blood Pressure Analysis System software.Mean SBP measurements were recorded weekly during the 10-week HFD regimen.

Fasted blood collection
Fasted bloods were collected fortnightly.Mice were fasted for 6 h (from 8:00 AM) with free access to water.Blood glucose was measured in samples from the saphenous vein using a handheld Accu-Chek® Guide glucose meter per manufacturer's instructions (NSW, Australia).Blood was collected (~ 120μL) for plasma isolation to measure insulin using a Mouse Ultrasensitive Insulin ELISA kit (ALPCO Diagnostics, Salem, NH, USA) as per manufacturer's instructions 29 .

Plasma estradiol measurement
Plasma steroids were extracted twice with diethyl ether to remove potential interfering substances 30 .The supernatant, containing extracted steroids, was collected, dried and reconstituted in phosphate buffer.Plasma was analyzed using an oestradiol antibody (ISWE0008, Arbor Assays, Ann Arbor, Michigan).Briefly, a 96-well flat-bottomed microtiter plate was coated with donkey anti-sheep immunoglobulin.The plate was washed and samples, standards (10-0.078ng/ml) and controls were loaded into each well, followed by the corresponding enzyme-labelled antibody.The plate was incubated for 2 h then washed.TMB substrate solution was added to each well.After 30 min, the reaction was stopped using 0.5 M H 2 SO 4 and the plate was read at 450 nm and 620 nm using the SPECTROstar Nano Plate reader (BMG Labtech, Mornington Victoria, Australia).All samples were analysed in duplicate.To assess matrix interference and determine the dilution factor for each assay, a parallelism between the standard curve and a serially diluted sample pool was performed for all samples.

Isolation of aortae
Mice were euthanized using carbon dioxide asphyxiation followed by cardiac puncture and injection of 0.1 mL of enoxaparin sodium (400 units; Clexane®, Auckland, NZ).Blood samples were measured for glycated hemoglobin (HbA1c), total cholesterol and triglycerides using the Roche Cobas b 101 Monitoring System (Basel, Switzerland 29 ).Mice were intracardially perfused with phosphate-buffered saline (PBS).Aortae were harvested, then placed in ice-cold PBS for either flow cytometry, histology or molecular analyses.Lymph nodes and excess visceral fat were carefully removed via microdissection 31

Flow cytometry
Flow cytometry was performed on cell suspensions derived from freshly isolated aortae (including perivascular adipose tissue) as previously described 31 .Aortae were minced with scissors and enzymatically digested in a buffer (MgCl 2 and CaCl 2 ) comprising a mixture of type XI collagenase (125 U/mL, C7657-100MG), hyaluronidase (600 U/mL, H3884-50MG) and type I-S collagenase (450 U/mL, C1639-50MG; all enzymes purchased from Sigma-Aldrich, USA) for 1 h at 37 °C, as previously described 31 .Samples were then passed through a sterile 70 μm filter (BD Biosciences, USA) and cells were pelleted by centrifugation at 1500 RPM for 5 min at 4 °C.Aortic cell suspensions were stained with an Aqua Live/dead cell viability stain (Invitrogen, USA; 1 in 1000 dilution in PBS) for 15 min at room temperature, followed by a master mix containing a cocktail of antibodies in MACS buffer (0.5% bovine serum albumin and 2 mM EDTA in PBS; full list of antibodies in Suppl Table 1).Lastly, stained cells were fixed in MACS buffer containing 1% formalin.All samples were analyzed using a CytoFLEX S analyser (Beckman Coulter, Indianapolis, IN, USA) using CytExpert software version 2.5 (Beckman Coulter, USA).Immune cell populations were quantified using FlowJo software (BD Biosciences, version 10.8.1; gating strategy in Suppl Fig. 1).Cell numbers were expressed as total cells per aorta.

Wire myography
Vascular function of the abdominal aorta was assessed as described previously 32 .Aortic rings (2 mm in length) were mounted on a 4-channel Mulvany-Halpern Myograph (model 610 M, Danish Myo Technology, Aarhus, Denmark), and allowed to stabilize at zero tension for 15 min followed by normalization and equilibration 33 .All experiments were performed in Krebs bicarbonate solution at 37 °C and bubbled with carbogen (95% O 2 and 5% CO 2 ).Changes in isometric tension were recorded using a Powerlab/Lab Chart data acquisition system (AD Instruments, Bella Vista, NSW, Australia).Vessels were maximally contracted with the thromboxane A 2 mimetic U46619 (1 μM bolus).This was followed by the determination of endothelial integrity as described previously 34,35 .To assess vasorelaxant function, aortic rings were pre-contracted to 70-80% of maximum U46619 contraction, using U46619, and cumulative concentration-response curves to the endothelium-dependent agonist acetylcholine (ACh; 0.1 nM-10 μM) or the endothelium-independent vasorelaxant NO donor sodium nitroprusside (SNP; 0.1 nM-10 μM) were performed, as previously described 33 .Additionally, responses to ACh and U46619 were examined after a 30 min incubation in the presence of a NO synthase inhibitor, Nω-nitro-ι-arginine methyl ester (L-NAME; 200 μM) or indomethacin (1 μM), a cyclooxygenase (COX) inhibitor.To assess this response, aortic rings were then pre-contracted to 50-70% of maximum U46619 contraction using U46619, and cumulative concentration-response curves to endothelial agonist acetylcholine (ACh; 0.1 nM-10 μM) were performed.Basal NO synthase reactivity was quantified through the addition of L-NAME (200 μM) to aortic rings sub-maximally contracted with U46619 to 20-30% of maximal U46619 contraction.

Histopathology
Aortic sections were fixed in 10% neutral buffered formalin, embedded in paraffin wax and cut into 4 µm sections.Vessels were stained with Masson's trichrome for histological analysis of collagen and adipocytes.Each whole slide was scanned using the Aperio Scanscope AT Turbo, and raw virtual slide files were viewed using Aperio ImageScope software (Leica Biosystems Microsystems, Mount Waverley, VIC, Australia) to extract the whole image.Collagen content and adipocyte cross-sectional area was quantified by a semi-automated macro in ImageJ (version 1.53t, National Institute of Health, USA).

Reagents
Drugs for wire myography were purchased from Sigma-Aldrich (St Louis, MO, USA), except for U46619 (Cayman Chemical, Ann Arbor, MI, USA).All drugs were dissolved in distilled water, except for indomethacin, which was dissolved in 0.1 mol/l sodium carbonate, and U46619, which was dissolved in 100% ethanol to create a stock solution at 1 mmol/l.All antibodies for flow cytometry were purchased from Biolegend, USA.

Statistical analysis
Data are expressed as mean ± SEM with n representing the number of mice per group.All data analysis was performed in GraphPad Prism (version 7.0, GraphPad Software, San Diego, CA, USA.Concentration-response curves from aorta were fit to a sigmoidal curve using nonlinear regression.ACh-and SNP-evoked relaxation with and without blockers was expressed as a percentage of pre-contraction evoked by U46619.Body weight, fasting blood glucose, plasma insulin, blood pressure and concentration-response curve data were analyzed using a two-way ANOVA, with Sidak's post hoc.Blood cholesterol, HbA1C, flow cytometry data, pEC50 and R max data were compared using a two-way ANOVA, with Tukey's post-hoc analysis.A level of P < 0.05 was considered significant.Power calculations indicated a minimum sample size of n = 7 for metabolic parameters, n = 5 for flow cytometry and n = 6 for wire myography was required to identify a 25% mean effect change in with 80% power, and standard deviation of 10%.Assessors and data collectors were blinded to experimental groups.

Results
HFD increased weight gain in both sexes, with sex-specific effects on metabolic parameters HFD increased body weight in both males and females compared to NCD mice (P < 0.05; Fig. 1a,b).Post hoc analyses revealed that in males, body weight was increased from week 5 (P < 0.05; Fig. 1a).Interestingly, the onset of obesity was delayed in females where HFD increased bodyweight in only weeks 9 and 10 of the diet regimen (P < 0.05; Fig. 1b).HFD induced hyperglycemia in both sexes, but at different timepoints.HFD increased fasting blood glucose in males (P = 0.001) from week 6 of the diet regiment (P < 0.05; Fig. 1c).In females however, HFD increased fasting blood glucose transiently at week 8 (P = 0.001; Fig. 1d).HFD increased fasting plasma insulin at week 10 in males but did not change in females (Fig. 1e,f) and SBP remained unchanged in both sexes (Fig. 1g,h).At the endpoint, HFD increased blood cholesterol in males (P = 0.03) but not females (Tables 1,  2).HFD increased plasma estradiol in males (P = 0.02) but not females (Tables 1, 2).HFD did not affect blood triglycerides or HbA1c in either sex when compared to NCD control mice (Tables 1, 2).

HFD-feeding impairs maximal endothelium-dependent vasorelaxation in males but not females
There were no differences in ACh concentration-response curves between diet groups in male or female mice (Fig. 4a,b, Tables 3, 4).However, there was an impaired maximal relaxation in males on HFD versus NCD (P = 0.003), but no diet effect in females (Tables 3, 4).To investigate the effect of obesity on the mechanisms of endothelium-dependent relaxation, concentration-response curves to ACh were evaluated in the presence of the NOS inhibitor, L-NAME or the COX inhibitor, indomethacin.There were no differences in ACh concentration-response curves between diet groups in the presence of either blocker (Fig. 4c-f).Area under the curve was calculated to determine contribution of NO and COX.HFD did not change the contributions of either mechanism to ACh-mediated relaxation of either sex (Fig. 4g,h).Similarly, HFD did not affect endotheliumindependent relaxation to the NO donor SNP, L-NAME-induced contraction, or half-maximal effective concentration to ACh (Fig. 5a-d, Tables 3, 4).

HFD increased adipocyte size in males with no changes in collagen
To assess the effect of HFD on aortic collagen content and perivascular adipocyte size, we performed Masson's trichrome staining.Aortic collagen content remained unchanged in response to HFD for both sexes (Fig. 6a-e), but HFD increased the mean area of aortic adipocytes in males (P = 0.003), but not females (Fig. 6a,c,f).

Discussion
Sexual dimorphisms in diet-induced models of obesity are understudied, with a bias towards studies in males.Our study addressed this gap by assessing vascular inflammation and function in male and female mice in response to the HFD.We showed that a 10-week HFD regimen induced obesity (increased body weight), hyperglycemia, hyperinsulinemia, and hypercholesterolemia in males.The development of these metabolic disturbances was blunted or absent in females.We also showed that HFD caused mild endothelial dysfunction in the aorta, but only in males.Conversely, HFD only altered aortic leukocytes in females -specifically, it resulted in fewer proinflammatory monocytes.
Our finding that males are more susceptible to HFD-induced metabolic disturbances than females is consistent with other studies that reported earlier HFD-induced weight gain in males than females 15,36,37 .Aligned with our findings, increased weight gain in males is evident after 5 weeks of HFD, but not until later in females 13 .While most studies have reported that excessive weight gain is blunted or delayed in females, one study reported similar weight gain between sexes after 14 weeks of HFD 38 .This difference could be attributed to the HFD being introduced to the mice at a later age (i.e.11-12 weeks of age) than most other studies 38 .Total cholesterol was increased in HFD males, with no changes in total triglycerides.This contrasts with females, where HFD had no effect on cholesterol or triglycerides.In other models of advanced HFD-induced obesity (i.e.12-32 weeks of HFD), plasma lipids and cholesterol were increased in both sexes 39 .Taken with our data, it is likely that dyslipidemia is delayed in females, rather than being completely absent.
HFD males had higher fasting blood glucose levels from week 6 and higher fasting plasma insulin levels from week 10 than NCD males.In contrast, HFD females only had a transiently higher fasting blood glucose at week 8, with no change in fasting plasma insulin.Consistent with other studies 15,36,38 , this suggests that insulin resistance occurs within 10 weeks of HFD in males, but not in females.The sexual dimorphism in insulin resistance is well-established, with males exhibiting a greater degree of insulin resistance compared to females.This disparity could be due to sex-specific traits, such as elevated visceral and hepatic adipose tissue, coupled with the absence of protective estrogen and lower adiponectin (an insulin-sensitizing hormone levels 40  www.nature.com/scientificreports/resistance occurring in males (and transiently in females), HFD did not affect glycated hemoglobin (HbA1c).This was expected, as blood glucose had been elevated for only a short period.HbA1c is an indicator of chronically increased blood glucose levels (i.e.≥ 3 months).Indeed, studies using longer HFD regimens (16-24 weeks) did report increases in both fasting blood glucose and HbA1c [41][42][43] .HFD increased plasma estradiol in males but not females.This was particularly interesting given the established protective effects of estrogen against obesity and metabolic complications in male mice 44 .Previous studies have reported that administration of estrogen in obese male mice was associated with improved weight gain and diet-induced metabolic changes 44 .However, the observed increase in estradiol in response to HFD is an endogenous response and corroborates with results in human males that also show elevated circulating estradiol in response to diets high in polyunsaturated fats whereby increased aromatase activity converts testosterone to estradiol 45,46 .Furthermore, the Barostoc feed used for NCD may contain isoflavones, a natural estrogenic compound found in legumes 47 .Consequently, the NCD groups may exhibit higher levels of plasma estradiol, which may mask a greater difference in plasma estradiol between NCD and HFD groups.One limitation of our study was that we did not account for the estrus cycle in female mice.There are studies that suggest that the rodent estrus cycle can have an impact on blood pressure, blood glucose, and estradiol levels 48,49 .By not including this, we may be potentially masking important differences between our experimental groups.Furthermore, we are also aware of the challenges associated with measuring plasma estradiol levels using direct immunoassays, especially at low concentrations 50,51 .However, the assays used were sensitive enough to detect estradiol levels in our study, and no significant differences in estradiol levels were observed between the groups.
Our flow cytometry data showed no effect of HFD on aortic immune cells in either sex.Surprisingly, we observed fewer Ly6CHi + proinflammatory monocytes in females on HFD compared to NCD.This suggests a female-specific compensatory mechanism to suppress aortic macrophages in response to HFD.This contrasts to other rodent HFD studies where HFD-induced obesity increased aortic inflammation in males -characterized by macrophage infiltration and secretion of inflammatory cytokines (interleukin-6, interleukin-8, tumor necrosis factor-α and monocyte chemoattractant-1) [52][53][54] .However, it is important to note that no previous studies included females and had a diet regimen longer than 10 weeks.
Aortic lymphocytes (T and B cells) remained unchanged in response to HFD.There were also no sex-specific responses in aortic lymphocytes.This was surprising as obesity-related chronic inflammation is typically associated with increased lymphocyte infiltration within adipose tissues 53 .However, previous HFD studies characterized T and B cells in visceral adipose tissues rather than the aorta (or aortic perivascular adipose tissue) and used longer diet regimens.Other HFD studies show increased CD8 + T cells and decreased CD4 + and regulatory T cells in the visceral adipose of obese males when compared to HFD females 52,54 .Moreover, Treg populations are typically reduced in visceral adipose tissue in mice fed a HFD 55,56 .To the best of our knowledge, no previous studies have characterized leukocytes in aortic perivascular adipose tissue in obese rodents.While our findings suggest that the typical changes to T cells do not occur in aortic perivascular adipose tissue in early obesity, more research is required to confirm this in models of advanced obesity.
Despite a slight reduction in the maximal vasorelaxation to ACh in HFD males, endothelial function was similar between HFD and NCD mice.This contrasts with other studies where HFD induced substantial endothelial dysfunction after ≥ 12 weeks 18,57,58 .Our 10-week HFD regimen was possibly not long enough to significantly reduce endothelial vasorelaxant function.Interestingly, one study reported that 8 weeks on a HFD increased aortic inducible NOS (iNOS) expression and NO production in male mice 59 .This suggests a short-term compensatory mechanism stimulated by inflammatory cytokines, however prolonged iNOS-derived NO can promote www.nature.com/scientificreports/endothelial dysfunction 59 .In our study of early obesity, males were more vulnerable to metabolic disturbances compared to HFD-females.However, previous studies in rodents on western diet for 16 weeks demonstrated sexual dimorphism in obesity-related endothelial stiffening 60 .This showed that females exhibit greater susceptibility to endothelial stiffening due to estrogen signaling when compared to males.This is observed in a clinical setting where diabetic women lose the cardiovascular protection of estrogen and consequently, exhibit higher incidence and severity of cardiovascular disease compared to men 60 .Therefore, our wire myography data, when combined with the observed metabolic disturbances, suggests that hyperglycaemia, hyperinsulinemia, and weight gain can precede endothelial dysfunction in the aorta in HFD-fed male mice.Endothelium-mediated relaxation in response to ACh when pharmacologically blocked with L-NAME or indomethacin was comparable between diet groups in both sexes.Therefore, we were unable to determine whether the mild impairment in maximal relaxation to ACh in HFD males was due to NO or prostanoids.Our AUC analyses revealed that aortic ACh-mediated relaxation predominantly occurred via NO, meaning it is more likely that impaired relaxation was due to a reduction of the NO contribution.Notably, prostanoids contribute significantly to ACh-induced relaxation in human saphenous veins and internal mammary arteries 61 .However, the contribution of prostanoids to ACh-induced relaxation in the human aorta has not been explored, therefore it is difficult to compare our findings to that in humans.Due to limited tissue availability, we were unable to further interrogate NO or prostanoid pathways.www.nature.com/scientificreports/Lastly, our data show that HFD resulted in larger aortic perivascular adipocytes in males but not females.While adipose tissue expansion is a well-recognized driver of inflammation, we did not observe aortic inflammation despite the increase in adipocyte size 62 .However, HFD males did exhibit hyperglycemia and hyperinsulinemia, consistent with adipocyte expansion accompanying metabolic disturbances but preceding inflammation.Moreover, unchanged adipocyte size coupled with fewer proinflammatory monocytes in HFD females suggests a protective mechanism exists within the perivascular adipose tissue of females, which may contribute to preserved endothelial function; however, this is a limitation of our study.Although perivascular adipose tissue was included in the flow cytometry studies, it was not included in the wire myography studies.Consequently, we were unable to examine the impact of HFD on perivascular adipose tissue and its secretory factors that could influence vascular function 63 .Additionally, further investigations characterizing aortic function, adipocyte size and inflammation and at different time points during a longer HFD diet study are required to clearly define the sequence of events occurring within the aorta.
While the primary objective of this study was to explore sex-specific responses to the HFD, the significance of understanding sex differences is also acknowledged.Unfortunately, the study was not powered to perform 3-way ANOVAs to investigate sex differences.This is a limitation of the study.Future studies should aim to focus on sex differences to gain a comprehensive understanding of the underlying biological mechanisms that contribute to the variation between males and females.Our study has characterized, for the first time, the early sex-specific effects of a HFD on metabolic parameters, vascular inflammation and vasorelaxant function in male and female mice.We identified that mild vascular dysfunction is a male-specific effect of HFD -likely influenced by the increased vulnerability to metabolic disturbances.Indeed, metabolic disturbances were blunted in HFD females and vascular function was preserved.Moreover, HFD reduced proinflammatory monocytes in females, which may have contributed to the protection from vascular dysfunction.Overall, our study highlights that sex-specific effects to HFD are apparent in the aorta during the early stages of obesity or pre-obesity, but further work is still needed to understand the implications of these differences in the later stages of disease. https://doi.org/10.1038/s41598-023-47903-1

Figure 5 .
Figure 5. High fat diet did not alter relaxation to sodium nitroprusside or basal NO.Male and female concentration-response curves to sodium nitroprusside (SNP; (a,b)) and maximal contraction to L-NAME (c,d) in C57BL/6 male (black) and female (red) mice fed a normal chow diet (NCD; filled circle & filled square) or high-fat diet (HFD; open circle & open square).Values are mean ± SEM, n = 6-7 per group.*P < 0.05 vs. NCD, 2-way ANOVA with Bonferroni's multiple comparison (a,b) or Student unpaired t test (c,d).

Figure 6 .
Figure 6.High fat diet increased aortic perivascular adipocyte size in male mice.Representative images of thoracic aorta stained with Masson's trichrome from (a) NCD male, (b) NCD female, (c) HFD male and (d) HFD female.Images were scanned at 10 × magnification.Collagen area (as a percentage of the total area of the vascular wall; (e) and average adipocyte cross sectional area (CSA; (f) in C57BL/6 male (black) and female (red) mice fed a NCD (filled circle & filled square) or HFD (open circle & open square; (e).Scale bar = 50 μm.Values are mean ± SEM, n = 5-10 per group.*P < 0.05 vs. NCD, for 2-way ANOVA with Sidak's multiple comparison test.

Table 3 .
The half-maximal effective concentration (pEC 50 ) and maximal relaxation (R max ) for acetylcholine (ACh) and sodium nitroprusside (SNP) in the aorta from male mice fed a normal control diet (NCD) or highfat diet (HFD).ACh concentration-response curves were performed in the absence or presence of L-NAME or indomethacin (Indo).Data are expressed as mean ± SEM, *P < 0.05 vs NCD.

Table 4 .
The half-maximal effective concentration (pEC 50 ) and maximal relaxation (R max ) for acetylcholine (ACh) and sodium nitroprusside (SNP) in the aorta from female mice fed a normal control diet (NCD) or high-fat diet (HFD).ACh concentration-response curves were performed in the absence or presence of L-NAME or indomethacin (Indo).Data are expressed as mean ± SEM.